The invention relates to devices and methods used in preparing gel strips for electrophoresis and performing electrophoresis on gel strips.
Two-dimensional electrophoresis is an effective way to analyze complex mixtures of proteins. Typically, two-dimensional electrophoresis involves separating the protein mixture by the intrinsic charge characteristics of the proteins, i.e., their isoelectric points, in a first dimension by a type of electrophoresis called isoelectric focusing, and then separating the protein mixture in a second dimension by electrophoresis. In the second-dimension electrophoresis, a gel strip containing the proteins separated in the first dimension is incubated in a buffer appropriate for the second-dimension electrophoresis, and applied to a second-dimension vertical or horizontal slab gel so that the proteins can be electrophoresed out of the first-dimension gel and into the second gel under appropriate conditions to separate the proteins on the basis of molecular mass.
The first-dimension electrophoresis, i.e., isoelectric focusing, is usually performed on thin flat strips of polyacrylamide gel containing a covalently immobilized pH gradient, i.e., IPG gel strips. The IPG gel strips are commercially available in a dehydrated state and are rehydrated in an appropriate buffer before use. Currently, each IPG gel strip is rehydrated in a first gel carrier apparatus, and then handled and transferred by the user to a second gel carrier apparatus for isoelectric focusing (IEF) to separate the supplied proteins by isoelectric point.
A problem with the present processes and gel carrier apparatuses for IPG gel strip preparation and isoelectric focusing is that the IPG gel strips tend to be fragile, flimsy, and difficult to handle between the steps of rehydration and first dimension electrophoresis, as is commonly practiced at present time. Transferring the gel strip from a first gel carrier apparatus for rehydration to a second gel carrier apparatus for isoelectric focusing requires to much handling and hands-on-time for the first-dimension electrophoresis.
To this end, a first aspect of the present invention involves a gel strip carrier module for a gel strip that reduces the handling of the gel strip and the hands-on-time during preparation of the gel strip for isoelectric focusing by including a gel strip chamber that serves as both a rehydration and focusing chamber.
A second aspect of the present invention involves a device for rehydrating and performing electrophoresis on a gel strip having two ends and a gel face. The device includes a holder having a gel strip chamber configured to receive the gel strip and a rehydration buffer medium. First and second electrodes are carried by the holder. The electrodes include internal electrical contact points adapted to contact the gel face of the gel strip near the ends of the gel strip and external electrical contact points adapted to be electrical connected to a power supply for performing electrophoresis on the gel strip.
A preferred embodiment of the above aspect of the invention includes a number of features. A first feature is that the internal electrical contact points are carried by the holder and are adapted to contact the face of the gel strip with the gel strip oriented gel face down in the chamber. A second feature is that the holder includes electrolytic gas bubble escape vents comprised of a widened area in the chamber adjacent to an internal electrical contact. A third feature is that the chamber may include an optional sample introduction area between the internal electrical contact points comprised of a widened area in the chamber adjacent to a lateral edge of the gel strip. A fourth feature is that the holder includes a floor and a wall, an inner surface of the wall and upper surface of the floor define the gel strip chamber, the gel strip chamber includes a first end and a second end, the internal electrical contact point of the first electrode is carried by the floor adjacent to the first end of the gel strip chamber, the internal electrical contact point of the second electrode is carried by the floor adjacent to the second end of the gel strip chamber. A sixth feature is that the gel carrier modules includes a cover for the gel strip chamber. A seventh feature is that at least one member is adapted to maintain the gel strip in contact with the electrical contacts, and the at least one member extends from the cover and includes a bottom surface and a height, whereby when the chamber is covered, the distance between the floor and the bottom surface of the at least one member is equal to the thickness of the gel strip when rehydrated. An eighth feature is that the gel carrier module may include a retaining mechanism adapted to retain the cover to the holder.
An alternative embodiment of the above aspect of the invention includes a number of features. A first feature is that the internal electrical contact points are carried by the cover and are adapted to contact the face of the gel strip with the gel strip oriented face up in the chamber. This embodiment of the invention may include a number of additional features. A second feature is that the holder includes a floor and a wall, an inner surface of the wall and upper surface of the floor define the gel strip chamber, the gel strip chamber includes a first end and a second end, the internal electrical contact point of the first electrode is carried by the cover configured to be adjacent to the first end of the gel strip chamber when the chamber is covered, the internal electrical contact point of the second electrode is carried by the cover and configured to be adjacent to the second end of the gel strip chamber when the chamber is covered. A third feature is that the cover includes a bottom surface, and the chamber includes a depth equal to the thickness of the gel strip when rehydrated so that the bottom surface of the cover maintains the gel strip in contact with the internal electrical contact points when the gel strip is rehydrated. A fourth feature is that the cover includes a sample loading reservoir. A fifth feature is that the cover includes a buffer introduction reservoir. A sixth feature is that the cover includes a bottom surface having a capillary break channel and an oil reservoir that communicates with the capillary break channel.
A third aspect of the invention involves a device for rehydrating and performing electrophoresis on a gel strip that includes a holder having a gel strip chamber configured to receive the gel strip and a rehydration buffer medium, and means for performing electrophoresis on the gel strip.
A preferred embodiment of the above aspect of the invention includes a number of features. A first feature is that the device includes means for venting electrolytic gas bubbles from the chamber. A second feature is that device includes means for introducing a test sample to the gel strip. A third feature is that the device includes means for retaining the cover to the holder. A fourth feature is that the device includes means for maintaining electrical contact between the gel strip and electrophoresis means. A fifth feature is that the device includes means for accommodating electroendosmotic flow in the gel strip during electrophoresis.
A fourth aspect of the invention involves a method of rehydrating and performing electrophoresis on a gel strip having first and second ends and a gel face. The method includes providing a device for rehydrating and performing electrophoresis on the gel strip comprising a holder including a gel strip chamber, the gel strip chamber configured to receive the gel strip and a rehydration buffer medium, and first and second electrodes having internal electrical contact points adapted to contact the gel face within the gel strip chamber near the first and second ends of the gel strip and external electrical contact points adapted to be electrically connected to a power supply for performing electrophoresis on the gel strip; providing a power supply; adding a rehydration buffer medium to the gel strip chamber; placing the gel strip into the chamber; and performing electrophoresis on the gel strip by applying voltage from the power supply to the external electrical contact points.
A preferred embodiment of the above aspect of the invention may include a number of features. A first feature is that the internal electrical contact points are adapted to contact the face of the gel strip with the gel strip oriented gel face down in the chamber, or the internal electrical contact points are adapted to contact the gel face of the gel strip with the gel strip oriented face up in the chamber. A second feature is that the gel strip may be placed into the chamber gel face down after the buffer medium is added to the rehydration chamber or gel face up before the buffer medium is added to the rehydration chamber. A third feature is that the method may include the step of providing an experimental protein sample. A fourth feature is that the chamber may include a sample introduction area between the internal electrical contact points so that the step of providing an experimental protein sample involves introducing the experimental protein sample in the sample introduction area along a lateral edge of the gel strip after the gel strip is lowered into the chamber, or alternatively, the experimental protein sample may be included in the rehydration buffer medium. A fifth feature is that the step of adding a rehydration buffer medium to the gel strip chamber occurs before placing the gel strip into the chamber, and the step of placing the gel strip into the chamber occurs before performing electrophoresis on the gel strip. A sixth feature is that the device includes a member adapted to maintain the gel strip in contact with the internal electrical contact points during electrophoresis of the gel strip. A seventh feature is that the device includes an electrolytic gas bubble escape vent that allows electrolytic gases produced at the internal electrical contact points during electrophoresis to escape. An eighth feature is that the device includes a cover for the gel strip chamber. Accordingly, the method further includes placing the cover on the gel strip chamber.
Other features and advantages of the inventions are set forth in the following detailed description and drawings, which are intended to illustrate, but not limit, the invention.